Chemoselective sequential ligation has been employed in applications including multiple labeling and syntheses of macromolecular assemblages. For examples of multiple labeling applications, see (a) Gramlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T. Angew. Chem. Int. Ed. 2008, 47, 3442; (b) Sanders, B. C.; Friscourt, F.; Ledin, P. A.; Mbua, N. E.; Arumugam, S.; Guo, J.; Boltje, T.; Popik, V. V.; Boons, G.-J. J. Am. Chem. Soc. 2011, 133, 949; and (c) Yi, L.; Sun, H.; Itzen, A.; Triola, G.; Waldmann, H.; Goody, R. S.; Wu, Y.-W. Angew. Chem. Int. Ed. 2011, 50, 8287. For examples of macromolecular assemblages applications, see (a) Durmaz, H.; Dag, A.; Altintas, O.; Erdogan, T.; Hizal, G.; Tunca, U. Macromolecules 2007, 40, 191; (b) Galibert, M.; Dumy, P.; Boturyn, D. Angew. Chem. Int. Ed. 2009, 48, 2576; (c) Kempe, K.; Hoogenboom, R.; Jaeger, M.; Schubert, U. S. Macromolecules 2011, 44, 6424; (d) Ehlers, I.; Maity, P.; Aubé, J.; König, B. Eur. J. Org. Chem. 2011, 2474; and (e) Valverde, I. E.; Lecaille, F.; Lalmanach, G.; Aucagne, V.; Delmas, A. F. Angew. Chem. Int. Ed. 2012, 51, 718. The two strategies in devising a sequential ligation are (1) successive coupling reactions of the same type that require the deprotection of the subsequent reactive site, and (2) the utilization of two or more completely different reactions. For examples of successive coupling reactions of the same type, see Gramlich et al., Ehlers et al., and Valverde et al. For examples of the utilization of two or more completely different reactions, see Sanders et al., Yi et al., Durmaz et al., Galibert et al., and Kempe et al. Recent research in bifunctional molecular linkers highlights the key challenge in developing chemoselective sequential ligation protocols, which is to achieve a complete regiochemical control of the coupling reactions in a timely and cost-effective manner, while providing broad functional group tolerance.
The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and its metal-free variants, which are the most widely applied “click” reactions, appear to be the methods of choice in developing bifunctional molecular linkers, due to their enormous substrate scopes and rapid reaction kinetics. See Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302; Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666; and Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004. Several examples of AAC-based bifunctional linkers known in the prior art are shown in the following Table 1 in which differentiating the reactivities of alkyne substrates leads to chemoselectivity in sequential ligation reactions.
TABLE 1Unsymmetrical Bisalkyne Linkers.CitationStructureAucagne and Leigh, 2006, compound 1 Girard et al., 2011, compound 2 Beal et al., 2012, compound 3
Aucagne and Leigh introduced a double-click method, in which two CuAAC reactions of compound I are spaced by a TMS-alkyne deprotection step to gain chemoselectivity. See Aucagne, V.; Leigh, D. A. Org. Lett. 2006, 8, 4505. In bisalkyne linker 2 by Girard et al., an electron-deficient propiolamide, which reacts with an azide thermally (i.e., without a copper catalyst), pairs up with a propargylic group targeted for a CuAAC reaction. See Elamari, H.; Meganem, F.; Herscovici, J.; Girard, C. Tetrahedron Lett. 2011, 52, 658. The pairing of cyclooctyne and terminal alkyne is utilized in compound 3 by Beal et al., in which the two triple bonds react via strain-promoted thermal reaction and copper(I)-catalyzed means, respectively. See Beal, D. M.; Albrow, V. E.; Burslem, G.; Hitchen, L.; Fernandes, C.; Lapthorn, C.; Roberts, L. R.; Selby, M. D.; Jones, L. H. Org. Biomol. Chem. 2012, 10, 548.
Although effective to various degrees, these earlier approaches suffer from a few limitations. The double-click method requires a protection/deprotection sequence, which adds workload. The thermal AAC reactions using strained or electron-deficient alkynes are relatively slow at rt, in addition to the lack of regioselectivity in affording 1,4- or 1,5-disubstituted triazoles. Furthermore, the propiolamide derivatives are prone to Michael addition with a nucleophile, thus limiting the scope of substrates in sequential ligations. In another noteworthy double-click method, amino-substituted organic azides are employed in which a diazo transfer reaction is required to activate the amino group to azido for the second CuAAC reaction. See Guiard, J.; Fiege, B.; Kitov, P. I.; Peters, T.; Bundle, D. R. Chem. Eur. J. 2011, 17, 7438.